Human breast cancer is the leading cause of cancer death in women from Western societies, and a large study of the epidemiology demonstrated strong associations between human prolactin and risk of breast cancer. Using established models of apoptosis of human breast cancer cell lines, we assessed the role of prolactin in breast cancer cell growth and survival. We showed that prolactin had no effect on the metabolic activity or total cell number of any cell lines. We confirmed endogenous prolactin production by these cells and that the levels varied. In the presence of a prolactin-neutralising antibody, each of the cell lines responded with the induction of apoptosis as opposed to growth inhibition. The sensitivity of the cell lines to the physiological inducer of apoptosis, C2-ceramide, appeared relative to the levels of endogenous prolactin that they contained. We then showed that exogenously added prolactin acted as a potent survival factor against apoptosis in all the cell lines examined. In addition, we demonstrated that a prolactin-neutralising antibody in combination with C2-ceramide caused an anticipated, additive increase in cell death. This study demonstrated that prolactin protects human breast cancer cell lines against apoptosis and this may have important implications for cancer treatment.